Viscous materials, such as gels and lipidic mesophases, including lipidic cubic phases (LCP), have been used successfully for the crystallization of proteins, including integral membrane proteins. The latter are typically very hydrophobic, and tend to aggregate amorphously instead of forming well-ordered three-dimensional crystals. In the practice of the LCP technique, a protein sample is mixed with a lipid to form a gel-like material of protein and lipid. The lipid portion of this material forms a cubic, three-dimensional, lattice which may promote formation of three-dimensional membrane protein crystals.
Crystallization conditions are not known initially, and so typically many crystallization experiments are prepared in an array format, such as in the wells of a 96-well plastic plate. A portion of prepared LCP material, containing the protein and lipid, is dispensed into each reservoir. The LCP material typically remains as a distinct, separate, phase from the aqueous crystallization solution during the crystallization experiment. Membrane protein crystals may be found within the LCP or in the dissolved lipid remnants. Protein crystals of soluble protein may be found anywhere within the crystallization space.
A problem associated with LCP-based crystallization experiments is that lipidic mesophase materials, and viscous LCP in particular, are difficult to dispense accurately. For example, a small portion of the LCP (e.g., 200 nL), is extruded with the aid of a syringe through a needle that is placed onto the surface of the well. The physical continuity of the LCP material has to be disrupted in order to dispense multiple portions of LCP material. In principle, this may be done, for example, by retracting the dispensation needle and thus breaking off a portion of the LCP. For this to occur the LCP has to be attached to a surface so that, when the dispensing needle is retracted, the LCP material adheres to the surface and a portion breaks off as the needle is withdrawn. Such strong bonding between the LCP and the surface material requires sufficient friction at the material interfaces. Sufficient friction is often not present, especially when the surface is not dry. Indeed, in order to prevent dehydration of the LCP material, the surface may be wet with a lubricating liquid. Even when LCP is dispensed onto a dry surface, slight dehydration of the LCP can induce lipid phase transition to form lamellar phases which create a lubricating layer between the surface and the bulk of the LCP material.
If the LCP does not break when the depositing needle is withdrawn, either no LCP material is deposited on the surface, or a variable amount of LCP is deposited in different wells, thereby introducing an additional variable in the protein crystallization experiment.